1. Field of the Invention
This invention relates to monoclonal antibodies, the hybridomas producing the antibodies, and methods for identifying and distinguishing vaccine strains of Newcastle disease virus from highly virulent Exotic Newcastle Disease virus. It also relates to peptides used to make the monoclonal antibodies.
2. Description of the Related Art
Newcastle disease virus (NDV), a member of the Paramyxoviridae family, is designated avian paramyxovirus 1. The enveloped virus has a negative-sense single-stranded genome of approximately 15 kb which codes for six proteins, including an RNA-directed RNA polymerase, hemaglutinin-neuraminidase protein, fusion protein, matrix protein, phosphoprotein, and nucleocapsid protein (Alexander, In: B. W. Calnek, H. J. Barnes, C. W. Beard, W. M. Reid, and H. W. Yoder, Jr (Eds), Diseases of Poultry, 9th ed., Iowa State University Press, Ames, Iowa, p. 496-519, 1991). Outbreaks of Newcastle disease were first reported for poultry from Java, Indonesia, and Newcastle-upon-Tyne, England in 1926. The disease currently has a worldwide distribution with a wide host range in which all orders of birds have been reported to be infected by NDV. Infectious virus may be ingested or inhaled, which results in its transmission and is the basis for mass-application vaccination procedures for poultry (Diseases of Poultry, 1991, supra). Isolates of NDV are categorized into three main pathotypes depending on the severity of disease produced by the isolate in chickens (Alexander, In: H. G. Purchase, L. H. Arp, C. H. Domermuth, and J. E. Pearson (Eds.), A Laboratory Manual for the Isolation and Identification of Avian Pathogens, 3rd edition, American Association of Avian Pathologists, Inc., Kennet Square, Pa.; 114-120, 1989; Alexander, 1991, supra). Lentogenic isolates do not usually cause disease in adult birds and are considered avirulent. Viruses of intermediate virulence that cause respiratory disease are termed mesogenic, while virulent viruses that cause high mortality are termed velogenic. In the United States, disease outbreaks of velogenic NDV in poultry are also termed Exotic New Castle Disease (END) and isolates from those outbreaks are termed END Virus (ENDV). Neurotropic and viscerotropic forms of velogenic viruses have been reported worldwide (Alexander, 1991, supra). Viscerotropic velogenic viruses have entered the United States via importation of psittacines (Bruning-Fann et al., J. Am. Vet. Med. Assoc., Volume 201, 1709-1714, 1992; Panigraphy et al., Avian Dis., Volume 37, 254-258, 1993; Senne et al., Avian Dis., Volume 27, 731-744, 1983) and were the causal agent of the major outbreak in southern California during the early 1970s (Schloer, ILnfect. Immun., Volume 10, 724732, 1968; Utterback and Schwartz, J. Am. Vet. Med. Assoc., Volume 163, 1080-1088, 1973). Outbreaks of Newcastle disease in cormorants in the north central United States and southern Canada in the 1990s were attributed to neurotropic velogenic viruses (Bannerjee et al., Avian Dis., Volume 38, 873-878, 1994; Wobeser et al., Can. Vet. J., Volume 34, 353-359, 1993).
Differential diagnosis of NDV involves hemagglutination inhibition with polyclonal NDV-specific antisera (Alexander, 1989, supra; Alexander, 1991, supra), or use of the enzyme-linked immunosorbent assay (ELISA) (Jestin et al., Arch. Virol., Volume 105, 199-208, 1989; Miers et al., Avian Pathol., Volume 27, 1112-1125, 1983; Wilson et al., Avian Dis., Volume 29, 1070-1085, 1984). Oligonucleotide probes (Jarecki-Black et al., Avian Dis., Volume 36, 134-138, 1992; Jarecki-Black et al., Avian Dis., Volume 37, 724-730, 1993) and viral genomic RNA fingerprint analysis (McMillian and Hanson, Avian Dis., Volume 26, 332-339, 1982; Palmieri and Mitchell, Avian Dis., Volume 35, 384-388, 1991) have been used to identify and differentiate NDV strains but with limited success. Monoclonal antibodies are now used to identify antigenic groups but pathotyping NDV isolates involves labor-intensive procedures. Pathotype prediction initially involves NDV inoculation of embryonated eggs to determine the mean time of death of the embryo. Further testing entails inoculation of chickens to determine the intracerebral pathogenicity index and the intravenous pathogenicity index. In the United States, the intracloacal inoculation pathogenicity test is used to distinguish viscerotropic velogenic NDV from neurotropic velogenic viruses. Additionally, virulent NDV can be differentiated by its ability to replicate in most avian and mammalian cell types without the addition of trypsin (Kaleta et al., Comp. Imm. Microbiol. Infect. Dis., Volume 2, 485-496, 1980; King, Avian Dis., Volume 37, 961-969, 1993; Nagai et al., Virology, Volume 72, 494-508, 1976). Although all NDV isolates can replicate in chicken embryo kidney cells, lentogenic viruses require trypsin for replication in avian fibroblasts or mammalian cells (King, supra).
Makkay et al. (Veterinary Microbiology, Volume 66 (3), 209-222, 1999) report that with the advent of subunit vaccines for microbial diseases, it is becoming increasingly important to be able to differentiate naturally infected animals from those vaccinated. Makkay et al. disclose the development of an Enzyme-Linked Immunosorbent Assay (ELISA) using baculovirus-expressed nucleocapsid proteins from Newcastle Disease virus as the coating antigen for a diagnostic and differential ELISA in order to detect antibody to subunit vaccines to Newcastle Disease Virus.
Roehrig et al. (Journal of Clinical Microbiology, Volume 29 (3), 630-631, 1991) disclose the preparation of a murine monoclonal antibody which differentiates between vaccine and wild-type Venezuelan equine encephalomyelitis viruses when used in an ELISA or an ImmunoFlourescent Assay (IFA). The reference discloses that the monoclonal antibody was derived from immunization with a synthetic peptide corresponding to the first 19 amino acids of the E2 glycoprotein of Trinidad donkey Venezuelan equine encephalomyelitis virus.
While methods are available for detecting the presence of NDV in animals, there remains a need for monoclonal antibodies, the hybridomas that produce them, and methods for differentiating high virulent Exotic Newcastle. Disease viruses from the low virulent NDV used to produce vaccines of the present invention which overcome some of the limitations of related art detection methods. The present invention described below are novel monoclonal antibodies, novel hybridomas producing the monoclonal antibody, novel peptide sequences used to produce the monoclonal antibodies and methods for using the antibodies and protein.